Interactions among filamentous fungi Aspergillus niger, Fusarium verticillioides and Clonostachys rosea: fungal biomass, diversity of secreted metabolites and fumonisin production

Abstract

Background: Interactions among fungi colonizing dead organic matter involve exploitation competition and interference competition. Major mechanism of interference competition is antibiosis caused by secreted secondary metabolites. The effect of competition on secondary metabolite production by fungi is however poorly understood. Fungal biomass was rarely monitored in interaction studies; it is not known whether dominance in pairwise interactions follows congruent patterns.

Results: Pairwise interactions of three fungal species with different life styles were studied. The saprophyte Aspergillus niger (A.n.), the plant pathogen Fusarium verticillioides (F.v.), and the mycoparasite Clonostachys rosea (C.r.) were grown in single and dual cultures in minimal medium with asparagine as nitrogen source. Competitive fitness shifted with time: in dual C.r./F.v. cultures after 10 d F.v. grew well while C.r. was suppressed; after 20 d C.r. recovered while F.v. became suppressed; and after 30 d most F.v. was destroyed. At certain time points fungal competitive fitness exhibited a rock-paper-scissors pattern: F.v. > A.n., A.n. > C.r., and C.r. > F.v. Most metabolites secreted to the medium at early stages in single and dual cultures were not found at later times. Many metabolites occurring in supernatants of single cultures were suppressed in dual cultures and many new metabolites not occurring in single cultures were found in dual cultures. A. niger showed the greatest ability to suppress the accumulation of metabolites produced by the other fungi. A. niger was also the species with the largest capacity of transforming metabolites produced by other fungi. Fumonisin production by F. verticillioides was suppressed in co-cultures with C. rosea but fumonisin B1 was not degraded by C. rosea nor did it affect the growth of C. rosea up to a concentration of 160 μg/ml.

Conclusions: Competitive fitness in pairwise interactions among fungi is incongruent, indicating that species-specific factors and/or effects are involved. Many metabolites secreted by fungi are catabolized by their producers at later growth stages. Diversity of metabolites accumulating in the medium is stimulated by fungus/fungus interactions. C. rosea suppresses the synthesis of fumonisins by F. verticillioides but does not degrade fumonisins.

Keywords: Fumonisin; Interference competition; Metabolic diversity; Metabolic profiling.

Fig. 1

Fungal biomass and metabolic diversity in single and dual cultures Aspergillus niger, Fusarium verticillioides, and Clonostachys rosea over time. Number of metabolic signals detected by HPLC-MS (grey lines) and the fungal biomass (bars) for single and dual cultures of Aspergillus niger, Fusarium verticillioides, and Clonostachys rosea after 10, 20, and 30 days of incubation are shown. Standard errors are indicated for the biomass in single cultures and for the number of metabolic signals. Standard errors for the biomass of fungi in dual cultures were smaller than 9 mg for all cultures and harvest time except A.n. in co-culture with F.v. at 20 d (13.6 mg) and C.r. in co-culture with F.v. at 30 d (10.2 mg)

Fig. 2

Comparison of metabolic profiles of single and dual cultures of Aspergillus niger, Fusarium verticillioides, and Clonostachys rosea. Cluster analysis was performed by UPGMA using the Jaccard’s similarity coefficients (coph. coeff. = 0.906) for HPLC-MS profiles of ethanol extracts of culture supernatants. Nodes denoted as ■ were supported by >75 % bootstraps for 2000 replicates. Single cultures: A - Aspergillus niger (A.n.); F - Fusarium verticillioides (F.v.); G - Clonostachys rosea (C.r.). Double cultures: AF - A.n./F.v.; AG - A.n./C.r.; FG - F.v./C.r. Incubation time (10, 20, and 30 days) is shown in the second part the labels of metabolic profiles

Fig. 3

Biotransformation of secreted fungal metabolites by mycelium of a different fungus. Ethyl acetate extracts of supernatants of 10 and 20 days old culture of one species were incubated with mycelium of a second species to determine whether the metabolites of one fungus can be transformed by another fungus. The resulting metabolic profiles were subjected to cluster analysis by UPGMA using Jaccard’s similarity coefficients (coph. coeff = 0.944). Nodes denoted as ■ were supported by >75 % bootstraps for 2000 replicates. A_Mycel, F_Mycel, G_Mycel: metabolic profiles of supernatants of washed mycelium of Aspergillus niger (A.n.), Fusarium verticillioides (F.v.), Clonostachys rosea (C.r.), respectively; A_Meta, F_Meta, G_Meta: metabolic profiles of supernatants of single cultures A.n., F.v., and C.r., respectively; X_Mycel + Y_Meta: metabolic profile of the supernatant after incubation of mycelium X with metabolites extracted from supernatant of Y

Fig. 4

Accumulation of fumonisins in pure cultures of F. verticilloides and dual cultures of F. verticillioides with C. rosea. The fungi were incubated in GM7 medium grown in the dark at 21 °C. Error bars show the standard error of mean. Concentrations of fumonisins in the supernatants in the course of time were determined by HPLC-MS/MS